ATPase Assay (written by: Danielle)
If you're reading this, I'll assume you have a basic understanding of the assay as described by Bryce here. Making Microtubules. You'll need to get unlabeled tubulin from the -80, You'll need 20uL of 25mg/mL unlabeled tubulin from row 4-2(last box). These tubes contain 40uL each, and one full tube usually gives me a concentration of about 100uM microtubules. You'll also need 100mM GTP, 2mM Taxol (you can make 2mM from 10mM by diluting in DMSO), 1M DTT and 20mg/mL BSA from the -30. *Most of these can be found in either the protein prep boxes, the pink TIRF box, or the ATPase assay box (bottom half, top right drawer). Additionally, you'll need to make either BRB80 or SRP with 1mM DTT, depending on which buffer your protein prefers. For this protocol, we'll use BRB80, but SRP can be used in each step in place of BRB80. Before starting, you should check to make sure that the TLA 100.1 rotor is at room temperature. If it is at 4C, you will need to heat it up on a 55C heat block. ***EVERYTHING AFTER STEP 3 IS AT ROOM TEMPERATURE*** # In each 20uL aliquot of tubulin, add 12uL BRB80(with DTT) # Add 8uL of 100mM GTP for a total of 40 (*NOTE your total should be 1/2 tubulin and 1/2 GTP+BRB80+DTT) # Place in the 37-degree water bath for a minimum of 15 minutes. # During this time, make 40uM Taxol (39.2uL BRB80+DTT and 0.8uL of 2mM Taxol) # After 15 minutes, take the tubes out of the 37 degree bath and add 40uL of 40uM Taxol to each tube, giving a total of 80uL per tube. # Place bath in 37 deg bath for a minimum of 15 mins. # During this time, make a 25% sucrose cushion: 200uL 50% sucrose(in PIPES-Hepes), 200uL BRB80+DTT, 2uL of 2mM Taxol. *Spin over 400uL Sucrose cushion (in larger tubes) # After 15 minutes, remove the tubes of tubulin from the 37 degree bath # In each TLA100 tube, add 400uL of sucrose cushion, and 80uL of your MTs. Be sure to create a balance using water. # ****CAREFULLY: layer your microtubules on TOP of the sucrose cushion. # Place each tube in the rotor, and spin in the common room (ultra centrifuge) at 70,000 RPM for 10 minutes # After spinning and before removing, mark the top of each tube so you know where the pellet should have formed. # Remove each tube and find the microtubule pellet(s). # After finding the pellet, remove the supernatant using a pipette, being very careful not to disturb the pellet # Resuspend each pellet in ~100uL of Assay Buffer with Taxol (see assay buffer below). # Measure the MT concentration by diluting 1uL in 4uL of Guanadine HCL, then measure the A280 (A280x5=__/116,000= __ x1,000,000 = Microtubule concentration in uM) Assay Reagents Assay Buffer (in 1mL total volume): 0.5uL of 20% Triton (totals 0.01% Triton) 1uL of 1M DTT (Totals 1mM DTT) 5uL of 20mg/mL BSA (Totals 0.1mg/mL BSA) 5uL of 2mM Taxol (Totals 10uM Taxol) BRB80 to 1mL total volume(*This could alternatively be SRP) 5mM NADH: 1mg NADH (in 4C deli fridge) 282uL H20 100mM PEP: 3mg PEP (in -30C Freezer inside ATPase Box) 133.4uL H20 11.6uL 5M KOH PK/LDH: Premade. You can find in ATPase box in -30C Assay Calculations Each assay has a standard equation, however, the best concentration of your ATPase and any inhibitors or enhancers of this ATPase must be determined. Here is the standard equation (volumes are in uL): * First, a concentration of ATPase (to use across all assays) must be determined. You should select a single concentration of MTs, say 1uM, and measure the activity at that specific concentration. Once you find an ATPase concentration that provides good stimulation at 1uM, then you can use this concentration for all assays.*NOTE: different preps of proteins may be different, so this will need to be done with each prep. * Next, a standard curve should be obtained at varying MT concentrations. Typically 0-8uM of MT is a range where you should be able to meet maximum activity. * After obtaining a standard curve, you may begin to add "effector" proteins you believe are inhibiting or enhancing the activity of your ATPase. To determine the best concentration to add, you will want to select a single concentration of MTs, say 1uM again, and also a single concentration of your ATPase, and vary the concentration of your effector protein. If you see stimulation or inhibition, depending on the literature on your two proteins of study, you will want to select the concentration where there is a drastic difference in stimulation of your ATPase, when compared to the standard curve at 1uM MTs. * To then explore the effect of this effector concentration, you can create another curve by varying MT concentrations and using the determined ATPase and effector protein concentrations you determined. * Once completed, you can compare this curve to the standard curve you've made and determine if there is a significant decrease or increase in the activity of your ATPase.